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UNIVERSITI PUTRA MALAYSIA
DETECTION OF ANTIBIOTIC RESIDUES AND ISOLATION OF ANTIBIOTIC RESISTANT ESCHERICHIA COLI FROM
CHICKEN MEAT AND CHICKENS IN MALAYSIA
TIN TIN MYAING
FPV 2003 17
DETECTION OF ANTIBIOTIC RESIDUES AND ISOLATION OF ANTIBIOTIC RESISTANT ESCHERICHIA COLI FROM
CmCKEN MEAT AND CHICKENS IN MALAYSIA
TIN TIN MYAING
DOCTOR OF PHILOSOPHY
UNIVERSITI PUTRA MALAYSIA
2003
DETECTION OF ANTmIOTIC RESIDUES AND ISOLATION OF ANTmIOTIC RESISTANT ESCHERICHIA COL/FROM
CHICKEN MEAT AND CHICKENS IN MALAYSIA
By
TIN TIN MYAING
Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia, in Fulfillment of the Requirement for the Degree of Doctor of Philosophy
January 2003
DEDICATED TO MY PARENTS,
MY HUSBAND AND MY SONS
II
Abstract of thesis submitted to the Senate of Universiti Putra Malaysia in fulfilment of the requirements for the degree of Doctor of Philosophy.
DETECTION OF ANTIBIOTIC RESIDUES AND ISOLATION OF ANTIBIOTIC RESISTANT ESCHERICHIA COLI FROM
CHICKEN MEAT AND CHICKENS IN MALAYSIA
By
TIN TIN MYAING
January 2003
Chairman: Associate Professor Dr. Saleha Abdul Aziz
Faculty: Veterinary Medicine
Public health is intrinsically related to food animal production. Antibiotic
residues in the food of animal origin and antibiotic resistant bacteria threaten human
health. There is an increase in population and the demand for chicken meat in
Malaysia is also increasing. Not much data is available on antibiotic residues and
antibiotic resistant E. eoll from chickens and foods of animal origin in Malaysia
In this study, a total of 400 chicken meat samples were subjected to
antibiotic residues screening tests. The prevalence of antibiotics residues in chicken
meat was between II 100 to 21 7%, using three microbial growth inhibition tests;
namely, fast antimicrobial screening test (FAST), Baedlus stearothermophdus disc
assay (BSDA) and a commercial test kit (TAT) with reference to four plate test
(FPT) The test performances were evaluated on sensitivity, specificity, positive
predtcnve value and negati\'e predictive value. The sensitivity of these tests ranged
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from 55.6 to 65% and specificity, from 82 to 90.6%. Kappa agreement was
between 0.5 to 0.8. Based on the above performance parameters, as well as the cost,
simplicity and incubation period, BSDA is a screening test of choice.
A total of 1 82 E. coli isolates from these chicken meat samples were found
resistant to twelve antibiotics; vancomycin (99.4%), trimethoprim (98.9%), nalidixic
acid (97.2%), tetracycline and cephradine (96.7%), ampicillin (94.5%), enrofloxacin
(83.5%), erythromycin (82.9%), ciprofloxacin (81 .3%), cefoperazone (80.2%),
chloramphenicol (74.4%) and kanamycin (68.6%). Forty-six antibiotypes and nine
antibiogroups were observed.
Escherichia coli isolated from antibiotic residues positive samples and
antibiotic residues negative samples were tested for antibiotic susceptibility to
twelve antibiotics. A higher percentage of antibiotic resistance was observed in E.
coli isolates from antibiotic residues positive samples compared to those from
antibiotic residues negative samples. 58.3% and 29.2% of E. coli isolates from
antibiotic residues positive samples and 25% and 17. 1 % of E. coli isolates from
antibiotic residues negative samples were resistant to 12 and 1 1 antibiotics,
respectively.
Plasmid isolation was conducted in 1 32 of the E. cob isolated. Plasmid
occurrence rate of 8 1 .8% were observed in this study with high diversity of plasmids
profiles among E. cob isolates from different sources. The number of plasm ids
ranged from 0 to 8 and the sizes of plasmids ranged from 1 .2 MDa to 1 18.6 MDa.
Forty-five different plasmid profiles were observed. No apparent correlation was
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found between the plasmid profiles of the strains and their antibiotic resistance
patterns.
In another study, the occurrence of antibiotic resistant E. coil was determined
In four flocks where in three flocks, chickens were given commercial feed
containing antibiotics and in one flock the feed given were without antibiotics.
Escherichia colt isolates from chickens given feed without antibiotics showed low
resistance to all antibiotics even at one day old. The screening of antibiotic residues
was done in 20 of these chickens at the age of 42 days old. The occurrence of
antibiotic residues was 10% in chickens given feed containing antibiotics. It was
observed that E. colt isolates from the antibiotic residues positive samples were also
resistant to 12 antibiotics while those from antibiotic residues negative samples were
resistant to 2-8 antibiotics. This observation requires further investigation.
Klebseilla spp., Pseudomonas spp. and E. coil were isolated from the feeds
and water of the flock where chickens given feed without antibiotics. All these
bacteria were resistant to 4-1 0 antibiotics. Antibiotic resistant E. coli was observed
in day-old chicks without any selective pressure, such as even when no antibiotic
was added to the ration. Thus, proliferation of antibiotic resistant E. colt is less
dependent on the use of antibiotics/antimicrobials In poultry farms and most likely
that chickens obtain antibiotic resistant E. colt from the environment.
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Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia bagi memenuhi syarat untuk ijazah Doktor Falsafah.
MENGESAN RESIDU ANTffiIOTIK DAN PENGASINGAN ESCHERICHIA COLI YANG TAHAN TERHADAP ANTffiIOTIK
DARIP ADA DAGING A YAM DAN A YAM DI MALAYSIA
Oleh
TIN TIN MYAING
Pengerusi: Profesor Madya Dr. Saleha Abdul Aziz
Fakulti: Perubatan Veterinar
Kesihatan awam berkait rapat dengan pengeluaran haiwan ternakan.
Kehadiran patogen bawaan makanan dan juga dalam makanan mengancam
kesihatan manusia. Terdapat peningkatan dalam populasi ayam dan permintaan
daging ayam juga meningkat. Data yang berkaitan dengan residu antibiotik and E.
coli tahan antibiotik dalam ayam adalah kurang di Malaysia.
Dalam kajian ini, sejumlah 400 sampel daging ayam telah diuji kehadiran
residu antibiotik. Prevalens residu antibiotik dalam daging ayam adalah antara
11.1 % hingga 21.7%, dengan menggunakan tiga ujian perencatan pertumbuhan
mikrobia, iaitu --fast antimicrobial screenmg test (FAST), Bacillus
stearothermoplulu.'i disc assay (BSDA) commercial test kit (TAT)" dengan dirujuk
kepada "four plate test (FPTf'. Prestasi ujian yang dinilai meliputi sensitiviti,
spesifisiti, nilai ramal an positif dan nilai ramalan negatif. Sensitiviti ujian tersebut
berkisar antara 55.6 hingga 65% dan spesifisiti daripada 82 hingga 90.6%.
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Persetujuan kappa adalah antara 0.5 hingga 0.8. Berdasarkan parameter prestasi
diatas, serta kos, kemudahan dan tempoh pengeraman, didapati BSDA adalah yang
terpilih sebagai ujian penyaringan.
Sejumlah 1 82 isolat E. cob yang diasingkan daripada sampel daging ayam
didapati tahan terhadap 1 2 jenis antibiotik- vankomisin (99.4%), trimetoprim
(98.9%), asid nalidik (97.2%), tetrasiklin dan sefaridin (96.7%), ampisilin (94.5%),
enrofloxasin (83.5%), eritromisin (82.9%), ciprofloxasin (81 .3%), cefoparazon
(80.2%), khlorampenikol (74.4%) dan kanamisin (68.6%). Terdapat 46 antibiotik
dan sembi Ian kumpulan antibio.
Eshenchla cob yang diasingkan daripada sampel yang positif dan yang
negatif residu untuk diuj i ketahanan terhadap dua belas jenis antibiotik. Keputusan
ujian menujukkan peratusan E. cob tahan antibiotik yang tinggi daripada sampel
yang positif untuk antibiotik berbanding dengan sampel yang negatif untuk residu
antibiotik. Terdapat 58.3% dan 29.2% E. cob daripada sampel positif untuk residu
antibiotik tahan terhadap 1 2 dan 1 1 antibiotik manakala 25% dan 17. 1 % E. cob
daripada sampel negatif untuk residu antibiotik yang tahan terhadap 1 2 dan 1 1
antibiotik.
Pengasingan plasmid dIlakukan ke atas 132 strain E. coli Kadar kehadiran
plasmid adalah 8 1 .8% dan terdapat kepelbagaian tlOggI dalam profil plasmid di
antara isolat E. cob yang diperoleh daripada peibagal sumber BIlangan plasmId
berkisar antara 0 hingga 8 dan saiz plasmid berada pada julat 1 2 MDa hingga 1 1 8 6
MDa. Empat puluh lima profil plasmid yang berbeza telah diperhatikan. Tlada
VI)
korelasi dijumpai antara profil plasmid strain E. coli dengan pola ketahanan
antibiotik.
Pada kajian selanjutnya, kewujudan E. coli tahan antibiotik ditentukan dalam
em pat kelompok ayam yang mana tiga kelompok ayam diberi makanan dengan
penambahan antibiotik dan dalam satu kumpulan makanan tiada penambahan
antibiotik. E. coil yang diasingkan daripada ayam yang diberi makanan tanpa
penambahan antibiotik menunjukkan ketahanan yang rendah terhadap semua
antibiotik, walaupun pada umur satu hari. Penyaringan residu antibiotik terhadap 20
sampel pada umur 42 hari telah dilakukan. Terdapat 10% positif untuk residu
antibiotik. Juga didapati bahawa E. coil yang diasingkan daripada sampel positif
untuk residu antibiotik didapati tahan terhadap 1 2 antibiotik, sedangkan ayam
daripada sampel negatif untuk residu antibiotik negatif tahan terhadap 2-8 antibiotik.
Pemerhatian ini perlu dikaji.
Klebsiella spp., Pseudomonas dan E. coli diasingkan dalam makanan dan air
daripada ayam diberi makanan tanpa penambahan antibiotik. Semua bakteria ini
didapati tahan terhadap 4-10 antibiotik. Esherzchla cob tahan antibiotik didapati
pada anak ayam umur satu hari, tanpa ada sebarang tekanan pemilihan seperti tiada
antibiotik ditambah pada makanan. Oleh itu, perkembang biakan E. coli tahan
antibiotik kurang bergantung pada penggunaan antibiotik dalam ladang ayam dan
diperkirakan bahawa ayam peroleh E. coli tahan antibiotik daripada persekitaran.
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ACKNOWLEDGEMENTS
First and foremost, my utmost appreciation, heartfelt thanks and gratitude
to Associate Professor Dr. Saleha Abdul Aziz, Chairman of Supervisory
Committee, for her kind guidance and close supervision that enable me to
complete my Ph.D study.
I would like to express my sincere gcatitude to Malaysian Technical
Cooperation Programme (MTCP) for the award of scholarship to pursue Ph.D
study in Universiti Putra Malaysia, Malaysia.
My special thanks to Associate Professor Dr. Raha Abdul Rahim,
Department of Biotechnology, Faculty of Food Science and Biotechnology,
Universiti Putra Malaysia, for her close supervision while doing molecular biology
work in her laboratory and Dr. Arifah Abdul Kadir also a member of The
Supervisory Committee, for her invaluable suggestions and comments.
My close appreciation and grateful to Brigadier General Maung Maung
Thein (Minister, Ministry of Livestock Breeding and Fishery), U Aung Thein
(Deputy Minister), U Kyaw Lwin (Director General), U Maung Maung Nyunt
(Director General), Dr. Maung Maung Sa (former Rector), Dr. Min Soe (former
Rector), Dr. Saw Plae Saw (former Pro-Rector), Dr. Tin Htwe (Pro-Rector) and Dr.
Yin Yin Thein (Assistant Director) for their kind encouragement and appreciation
through out my studies.
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My sincere thanks to Dr. Maznah Ahmad (Director), Puan Mamie and
Puan Norzila, in Veterinary Public Health Diagnostic Laboratory, Petaling Jaya,
for their generous support while doing my research work in the laboratory.
My special thanks to Dr. Ganapathy Kannan, for his guidance and kindness
in the samples collection from the poultry farms.
I am grateful to Dr. Zulkifli Idrus, Department of Animal Science, Faculty
of Agriculture, Universiti Putra Malaysia, for his kind permission to use the
poultry experimental facilities.
My special and sincere thanks to Encik Kamarzaman Ahmad in Veterinary
Public Health Laboratory, Faculty of Veterinary Medicine, who willingly assist
me in my works.
I am also grateful to Encik Zaid Othman and Encik Ghazali Md. Yusoff for
providing the transport to various places in samples collection.
My sincere thanks to all colleagues and staff of Faculty of Veterinary
Medicine, Universiti Putra Malaysia, for their contribution in the completion of
my study.
My heartfelt and special thanks to Dr. Myint Thein (Faculty of Veterinary
Medicine), U Win Myint (Faculty of Forestry), Dr. Kyaw Zay Ya (Faculty of Food
Science and Biotechnology), Dr. Than Da Min (Faculty of Agriculture), Dr. Nwe
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Nwe Htin (Faculty of Agriculture) and Miss Chua Lee Chuan (Faculty of Education)
for their kind moral support and encouragement.
Finally, my heartfelt thanks and appreciation to my husband Professor Dr.
Aye Cho, my sons, Zayar Aye Cho, Tay Zar Aye Cho and Yar Zar Aye Cho for
their love, support and encouragement to strive harder.
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I certify that an Examination Committee met on the 16th of January 2003 to conduct the final examination of Tin Tin Myaing on her Doctor of Philosophy thesis entitled "Detection of Antibiotic Residues and Isolation of Antibiotic Resistant Escherichia coil from Chicken Meat and Chickens in Malaysia" in accordance with Universiti Pertanian Malaysia (Higher Degree) Act 1980 and Universiti Pertanian Malaysia (Higher Degree) Regulations 1 981. The Committee recommends that the candidate be awarded the relevant degree. Members of the Examination committee are as follows:
ABDUL RANI BAHAMAN, Ph.D. Professor, Faculty of Veterinary Medicine, Universiti Putra Malaysia. (Chairman)
SALEHA ABDUL AZIZ, Ph.D. Associate Professor, Faculty of Veterinary Medicine, Universiti Putra Malaysia. (Member)
RAHA ABDUL RAHIM, Ph.D. Associate Professor, Department of Biotechnology, Faculty of Food Science and Biotechnology, Universiti Putra Malaysia. (Member)
ARIF AH ABDUL DAKIR, Ph.D. Lecturer, Faculty of Veterinary Medicine, Universiti Putra Malaysia. (Member)
CARLTON LLOYD GYLES, Ph.D. Professor, Department of Pathobiology, Ontario Vetennary College, University of Guelph, Canada (Independent ExamIner)
HAMSHER MOHAMAD RAMADILI, Ph.D Professor I Deputy Dean School of Graduate Studies, Umversiti Putra Malaysia.
Date 2 2 J.\.�� 70Cl
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This thesis submitted to the Senate of Universiti Putra Malaysia has been accepted as fulfilment of the requirements for the degree of Doctor of Philosophy. The members of the Supervisory Committee are as follows:
SALEHA ABDUL AZIZ, Ph.D. Associate Professor, Faculty of Veterinary Medicine, Universiti Putra Malaysia. (Chairperson)
RAHA ABDUL RAHIM, Ph.D. Associate Professor, Department of Biotechnology, Faculty of Food Science and Biotechnology, Universiti Putra Malaysia. (Member)
ARIF AH ABDUL DAKIR, Ph.D. Lecturer, Faculty of Veterinary Medicine, Universiti Putra Malaysia. (Member)
AINI IDERIS, Ph.D, Professor / Dean School of Graduate Studies Universiti Putra Malaysia
Date:
XIIl
DECLARATION
I hereby declare that the thesis is based on my original work except for quotations and citations, which have been duly, acknowledge. I also declare that it has not been previously or concurrently submitted for any other degree at Universiti Putra Malaysia or other institutions.
Tin Tin Myaing
XIV
TABLE OF CONTENTS
Page
DEDICATION.. . .. ....... ...... ..... ..... ..... ......... .............. ........... .... ..... ............ .......... 11 ABSTRACT.. .. . . . ..... . . . . . . ... . . ..... . . . .. . . . . . . ........... . . . . . . . . . . . . .. . . . . . . ... . . .. . . .. . . ......... ..... . . .. . 111 ABSTRAK. ..... . . . . . .... . . . . ... . ... . .. . . . . . . . .. .. .. . ... . . . ...... . . . . . .. . . . . . . . . .. . ...... . . . . . . . . . . ... . . . ... . . ... VI ACKNOWLEDGEMENTS . .. . . . . . .. .. . . ... . . .. . .. . . . . . . . . .. . . . . . .. . . . . ..... . . . . . . . .. .. . . .. . . . . . . . . . . . . IX APPROVAL S HEETS. . . . .. . . . . . . . ... ..................................................... ................. XlI DECLARATION . . . . . .. . .... . . . . . .. . .. . . . . . . . .. . ..... . . . . .. . . . . . . . . .. . . . . . .. ... . ..... . .... . .......... . . . . . . . . XIV LIST OF TABLES ...... . . . . . . . ... . . . . . .. . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . .. . .... . ............................. XIX LIST OF FIGURES . .... ... . . . ... . .. .. .. .... . . . . .. . . . ... . . . . . . . . . . . . . . ... . . . .. . .. .... . . .. .. . . . . . . .... . . . . . . . XXIV LIST OF ABBREVIATIONS . . .. . . . . ... . .... . . . . . . .. .. . . . . . . . .. ... . . . . . .. . . .. ...... ... . . ... . . . . . . ..... XXIX
CHAPTER
I
n
INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... . . . . . . . . . . . . . 1
Objectives of the Present Study . .... . ........ ... . . . .. . .. . . ........ 5
LITERATURE REVIEW . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Antibiotic and Antibiotic Residues in Farm 6 Animals . . . .. . . .. . . . . . . .. . . . . . . . . .... . .. .. . . . . . . . ........ ..... . . .. . . .. . . . . . . . . . .
The Use of Antibiotic in Farm Animals ... ....... 6 The Aim of Antibiotic Use in Farm Animals... . 7 Benefits and Risks of Antibiotic Use in Farm Animals ... . . . . . . . . . . . ..... .. . . . .. . . . . . .. .. . . . ... ...... .. . . . . ... . .. . 7 Mode of Action of Antibiotics ...... ................... 9 The Occurrence of Antibiotic Residues in Food of Animal Origin. . . .. .... ... . .. .. . ......... . . .. .. . .. . . 1 1 Public Health Aspects of Antibiotic Residues in Food of Animal Origin . ... . .. . . . . . . . . . . . . . . . . . . . .. . . . . . 13 Screening Methods for Detection of Antibiotic Residues. . . . . . .. . . . . ...... ............ .............................. 15 Maximum Residues Limit (MRLs) . . . . .. . ...... . . . . 16
Escherichia coli............................................................. 17 Taxonomy of Escherichia coli . . . . . . .. . . . . . . . . . . . . . . . . . 17 Typing Schemes of Escherichia coli .. . . .. . . . . . . . . 19 Prevalence of Escherichia coli . ... ..... . .. ... . . ....... 19 Public Health Significance of Poultry Meat Contaminated with Escherichia coli . . . . . . . . . . . .. . . 21
xv
m
Page
Antibiotic Resistance Pattern and Plasmid Profile................. 24 Nature of Antibiotic Resistant Gene........... 25 Transfer of Antibiotic Resistance............... 26 Transfer of Antibiotic Resistant Gene........ 27 Plasm ids. ..................................... .............. 29
Antibiotic Resistance Mechanism.......................................... 30 Resistance to j3-1actam Antibiotics.... ... ................. 31 Reistance to Quinolones ............ ........................... 31 Resistance to Tetracycline..................................... 32 Resistance to Aminoglycosides................. ............ 33 Resistance to Glycopeptides Antibiotics .............. 33 Resistance to Macrolides and Lincosamide........... 33 Resistance to Trimethoprim and Sulfonamides..... 34
Relationship between Antibiotic Residues and Antibiotic Resistant Organisms .............. ............................ 34
DETECTION OF ANTIBIOTIC RESIDUES IN CmCKEN MEAT AND EVALUATION OF SCREENING METHODS ..... . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . 36
Introduction ........................................................................ 36 Epidemiological Considerations for Effective Interpretation of Test Results.............................................. 38 Objectives of This Study .................................................. .. 39 Materials and Methods ................................... .................... 39
Samples Collection ..... ................. ...... .... ............... 39 Antibiotic Residue Screening Methods ................. 39 Four Plate Test (FPT) ............................................. 40 Fast Antimicrobial Screening Test (FAST) ........... 40 Bacillus stearothermophilus Disc Assay (BSDA).. 41 A Commercial Antibiotic Test Kit (TAT).............. 42 Identification of Tetracycline Residues by ELISA. 43 Test Interpretation of the Four Screening Tests... ... 43 Test Agreement and Decision Making........... ........ 45
Results ................................................................................ 46 Test agreement between FPT and FAST................ 52 Test agreement between FPT and BSDA............... 54 Test agreement between FPT and TAT.................. 55
Discussion ... ..... .... ............................................................... 69
XVI
IV
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Page
ANTffiIOTIC RESISTANT PATTERN OF ESCHERICHIA COLI ............................................ . 79
Introduction...................... ............. ............ ................... 79 Objectives of This Study.. ....... ... .... ..... ...... ......... .......... 82 Materials and Methods .. ............ ................................ .. 83
Bacterial Strains . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . 83 Isolation of Escherichia coli . . . . . . . . . . . . . . . . . . . . . . . . . . . 83 Antibiotic Sensitivity Test . . . . ... . . . . . . . . . . . . . . . . . . . . . . . 83 Antibiotic Disc .... ........ ....... .... ........................ 84 Interpretation of Inhibitory Zone.. ............... .... 84 Multiple Antibiotic Resistance Indexing of Isolates (MAR) ..... ..... .... ... ... . . . . . . . . . . . . . . . . . . . . 84 Detection of Antibiotic Residues and percentage of Antibiotic Resistant Escherichia coli. . . . . . . . . . . ... ............. . . . . . . . . . . . . . . . . . . . . . 85
Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85 Discussion ................... .. ... .... ......... .......... .... ............... 105
PLASMID PROFILES OF ESCHERICHIA COLI ISOLATES FROM CHICKEN MEAT . . . . . . . 1 1 5
Introduction . .. ... ..... ..... ............ ................. ................... 1 1 5 Objectives of This Study..... .. ...... ...... ...... ....... ........ ..... 1 17 Materials and Methods............ ..... .... ... .. ...... ............... 1 17
Sample Collection.. ....... .... ..... ... ... ................. .. . 1 17 Bacterial Isolation . . . . . . . . . . . . . . ... . . . . . . . . . . . . . . . . . . . . . . . . . . 1 17 Antibiotic Susceptibility Test . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1 7 Plasmid Isolation . ... .. ... ...... . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1 8 The Modified Alkaline-LysisIPFGE precipitation Procedure ...... ............................. 1 1 8 Agarose Gel Electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 20 Determination of Molecular Weight of Plasmid............................................................. 121
Results.......................................................................... 123 Correlation between Antibiotic Resistance and 147 Plasmid carriage among Escherichia coh Isolates ................................................................ .
Discussion. ..... ..... ... ........ ................. ... ...... ...... ............ ..... 152
XVII
VI
VII
COMPARISON OF ANTmIOTIC RESISTANT ESCHERICHIA COLI ISOLATED FROM CmCKENS GIVEN FEED WITH AND WITHOUT ANTffiIOTICS .... ..... ... ..... ..... ..... .... ... . .. . .
Page
1 56
Introduction .... ... ...... ... ... ... ... ... ... .... . . ....... ... .... ..... .. .... ... 1 56 Objectives of This Study .... ... ... ... .. .. ..... ..... ............. .. ... 1 58 Materials and Methods... .. .... .. .... .. ... .... ... .............. .... .. . 1 59
Sample Collection . ....... ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 59 Bacterial Isolation and Identification ... .. .... .... 1 59 Antibiotic Susceptibility Test . .. . . . ... .. . . . .... .. . .. . 1 59 Control Farm (Flock D) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 60 Isolation of Escherichia coli from Feeds and Water in Control Farm (Flock D) . . . . . . . . . . . . . . . . . . . 1 60 Statistical Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 6 1
Results . .. .. . .. .. . ... . . .. .. . .. .. .... ....... .. . .... .. .... ....... .... . ... ... . . . .. .. 16 1 Discussion . . .. ... . . . . ... . . .. . . . . ..... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223
GENERAL DISCUSSION AND CONCLUSION ..... 228
BmLIOGRAPHY.... . . . . . . .. . . . .. . . . . . . . . .... . . ......... ... . ... ..... ... .. .... . . . . . . ......... ....... 237
APPENDIX.. . . . . . . . . . . . . . . . . . . . . . . .. .. . ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265
VITA.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 266
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LIST OF TABLES
Table Page
1 Impression of antibiotic use in humans and animals.................... 7
2 Mode of action of antibiotics........................................................ 9
3 Occurrence of antibiotic residues in farm animals........................ 20
4 Biochemical tests for identification of Escherichia coli. . . ............. 1 8
5 Serogroups and disease associations of six virulent types of Escherichia coli. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...... 22
6 Characteristic of Escherichia coli- related illness in human....... 23
7 Evaluation of a test using a 2 x 2 table . . ........ .......................... .... 44
8 Kappa agreement............................................................................ 46
9 Comparison in performance parameters of FAST, BSDA and TAT standardized against FPT. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ....... 50
1 0 Test performance ofFPT against ELISA......... .............................. 5 1
1 1 Test performance of FAST against FPT. . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
12 Test performance of FAST against FPT at (95%) confidence interval............................................................................................ 53
1 3 Test performance of BSDA against FPT. . .. . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . .... 53
14 Tests performance ofBSDA against FPT at (95%) confidence intervaL.................. .. . . . . . . . .. . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . 54
1 5 Test performance of TAT against FPT........................................... 55
16 Tests performance of TAT against FPT at (95%) confidence intervaL................................................................................. . . . . . . . . . . . 56
17 Comparison in test performance of FAST, BSDA and TAT against FPT at (95%) confidence intervaL................................... 56
XIX.
18 Tests performance ofFP T, FA ST, BSDA and TA T at (95%) confidence interval......................................................................... 59
19 Relationship between prevalence and predictive value of FA ST............................................................................................. 63
20 Relationship between prevalence and predictive value of BSDA ................ ....... . . . . ....................... ....... ................................... 64
21 Relationship between prevalence and predictive value of TA T....................... ... . ... ........ . ..... .... ............................................... 65
22 Comparison of bacterial inhibition test........................................ 67
23 Different parameters ofFPT, FA ST, BSDA and TA T................. 68
24 Number and percentage of Escherichia coli isolates resistant to individual antibiotics .................................................. .............. 88
25 Percentage of Escherichia coli isolates resistant to a number of antibiotics ..................................................................................... 91
26 MAR index of Escherichia coli isolates ........................................ 93
27 Antibiogramme of Escherichia coli isolates from chicken meat.... 96
28 Antibiogroups of Escherichia coli isolates from chicken meat ..... 98
29 Comparison of Escherichia coli isolates from antibiotic residue positive and antibiotic residue negative samples resistant to individual antibiotic.. ......... ............................................................. 100
30 Comparison of Escherichia coli isolates from antibiotic residue positive and antibiotic residue negative samples resistant to number of antibiotic........................................................................ 103
31 Percentage of Escherichia coli isolates resistant to individual antibiotics ....................................................................................... 124
32 Plasmid DNA fragments harboured by Escherichia coli isolates 126
33 Plasmid profiles and antibiogramme of Escherichia coli .... .... ..... 143
34 Antibiotic resistance pattern of tscherichlO coli isolates without plasmid.......................................................................... 148
35 Resistant to antibiotics among Escherichia coli isolates with and without plasmids ............................................................. , ... ............ 149
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Number and percentage of plasm ids harboured by Eschenchta cob isolates in relation to number of antibiotics they are resistant to ................................ .. .. ... ... . . ... .. . . . . ... . .. ................. . .. .
Occurrence of Eschenchta coil isolates in one- day - old chicks, 21 days old and 42 days old chickens in flocks A, B, C and D ....
Percentage of Eschenchta cob isolates resistant to individual antibiotic at different ages in flock A ......................................... .
Antibiotic resistance pattern of Eschenchta coil isolated from one - day - old chicks, 21 days old and 42 days old chickens in flock A .. .... . ................................................ ............................. . . . . .
Antibiogroups of Eschenchia coil isolates in flock A ............ .
Eschenchta coil isolates resistant to number of antibiotics at the age of one - day - old chicks, 21 days and 42 days old chickens in flock A ..................................................................................... .
Percentage of Eschenchta coil isolates resistant to individual antibiotic at different ages in flock B .......................................... .
Antibiotic resistance pattern of Eschenchta coil isolated from one - day - old chicks, 21 days old and 42 two days old chickens in flock B ..................................................................... .
Antibiogroups of Eschenchta coil isolates in flock B ............. .
Eschenchta coil isolates resistant to number of antibiotics at the age of one - day - old chicks, 21 days old and 42 days old chickens in flock B ...................... ............................................... .
Percentage of EscherlChta coil isolates resistant to individual antibIOtic at different ages in flock C ..... ................................... .
Antibiotic resistance pattern of Eschenchta coil isolated from 21 days old and 42 days old chickens in flock C ....................... .
Antibiogroups of Eschenchta colt isolates in flock C ............ .
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Escherichia coli isolates resistant to number of antibiotics at the age of 21 days and 42 days old chickens in flock C .................. .
Percentage of Escherichia coli isolates resistant to individual antibiotic at different ages in flock D .......................................... .
Antibiotic resistance pattern of Escherichia coli isolated from one - day - old chicks, 21 days old and 42 days old chickens in
flock D ......................................................................................... .
Antibiogroups of Escherichia coli isolates in flock D .............. .
Escherichia coli isolates resistant to number of antibiotics at the age of one - day - old chicks, 21 days and 42 days old chickens in flock D ...................................................................................... .
Total number and percentage of Escherichia coli isolates resistant to individual antibiotic in one - day - old chicks in flocks A, B, C and D ................................................................... .
Total number and percentage of Escherichia coli isolates resistant to individual antibiotic isolated from 21 days old chickens in flocks A, B, C and D ............................................... .
Total number and percentage of Escherichia coli isolates resistant to individual antibiotic isolated from 42 days old chickens in flocks A, B, C and D ................................................ .
Comparison of Escherichia coli isolates resistant to number of antibiotics isolated from one - day - old chicks in flocks A, B, C and D ........................................................................................... .
Comparison of Escherichia coli isolates resistant to number of antibiotics isolated from 21 days old chickens in flocks A, B, C and D . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Com pal ison of Escherichia coil isolates resistant to number of antibiotics isolated from 42 days old chickens in flocks A, B, C and D ............................................................................................. .
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Antibiotic residues positive and negative samples from flocks A, B, C andD ............................................................................. .
Antibiotic resistance pattern and antibiotic residues positive and negative results of Escherichia coli isolates in 42 days old chickens in flocks A, B, C, and D ............................................... .
Antibiogramme of Escherichia coli isolated from antibiotic residues positive and negative samples in 42 days old chickens in flocks A, B, C and D ............................................................... .
Mean value of antibiotic resistant Escherichia coli isolates in one - day - old chicks in flocks A, B, C and D using Duncan's multiple range test ...................................................................... .
Mean value of antibiotic resistant Escherichia coli isolates in 21 days old chickens in flocks A, B, C and D using Duncan's multiple range test. ....................................................................... .
Mean value of antibiotic resistant Escherichia coli isolates in 42 days old chickens in flocks A, B, C and D using Duncan's multiple range test. ....................................................................... .
Antibiotic susceptibility test of Klebseilla isolated from feed in flockD . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . ... . .. .. . . . . . . .. . . . . .. . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . .
Antibiotic susceptibility test of Pseudomonas isolated from feed in flock D ..................................................................................... .
Antibiotic susceptibility test of Escherichia coli isolated from drinking water in flock D .......................................................... .
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